Project 1: Deciphering Mechanisms for Triglyceride and Cholesterol Transport SUMMARY/ABSTRACT Project 1 investigators have devoted their careers to exploring basic mechanisms of lipoprotein metabolism in health and disease. They discovered that an endothelial cell protein, GPIHBP1, is responsible for transporting lipoprotein lipase (LPL) to the capillary lumen; that the LPL?GPIHBP1 complex is crucial for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries; and that GPIHBP1 protects LPL from spontaneous and ANGPTL4-catalyzed unfolding/inactivation. Their efforts have resulted in >60 publications, many reflecting a commitment to understanding human disease. For example, they identified GPIHBP1 mutations causing chylomicronemia and uncovered a new human disease?chylomicronemia from GPIHBP1 autoantibodies. Recently, Project 1 investigators and coworkers determined the structure of the LPL?GPIHBP1 complex. During the next 5 years, Project 1 investigators will pursue two independent objectives. The first is to pursue ongoing studies of intravascular lipolysis, building on insights from the structure of the GPIHBP1?LPL complex. That structure, along with new reagents, new methodologies, and expert collaborators, have made intravascular lipolysis more exciting than ever. Key goals include defining amino acid residues required for GPIHBP1?LPL interactions, exploring mechanisms underlying specific ?chylomicronemia mutations,? understanding a gain-of- function polymorphism in LPL, defining the role of GPIHBP1?s acidic domain in stabilizing LPL from unfolding/inactivation, examining the function of GPIHBP1?s acidic domain in recruiting LPL from heparan sulfate proteoglycan binding sites in the subendothelial spaces, and investigating how ANGPTL4 initiates the unfolding and inactivation of LPL. We will also determine the structure of GPIHBP1 and LPL in association with an Fab fragment of the LPL?specific monoclonal antibody 5D2. Our second objective is to investigate the distribution of cholesterol in macrophages and the mechanisms by which macrophages dispose of cholesterol. In preliminary studies, Project 1 investigators found that macrophages release, by plasma membrane budding, numerous 30? 70-nm particles. By NanoSIMS imaging, these particles are highly enriched in cholesterol, including the metabolically active ?accessible cholesterol? detectable by bacterial cytolysins (e.g., ALO-D4). The finding that cholesterol-rich particles ?bud? from macrophages raises many questions. What is the function of particle budding? What is the composition of these particles? Is particle budding regulated? In collaboration with projects 2 and 3, project 1 will investigate the numbers and composition of macrophage particles in different settings (e.g., sterol starvation, cholesterol loading, LXR agonist treatment, and deficiencies of LXRs, ABCA1, or ABCG1). Preliminary NanoSIMS imaging studies showed that high-density lipoproteins are effective in unloading cholesterol from macrophage-derived particles, implying that macrophage particle budding could be relevant to reverse cholesterol transport and the emergence of cholesterol-laden cells in atherosclerotic plaques.